Characterization of the integration and modular excision of the integrative conjugative element PAISt in Streptomyces turgidiscabies Car8

Abstract

PAISt is a large genomic island located in the chromosome of the plant pathogen Streptomyces turgidiscabies Car8. The island carries clustered virulence genes, transfers to other Streptomyces species, and integrates by site-specific recombination at the 8 bp palindrome TTCATGAA. The palindrome is located at the 3' end of the bacitracin resistance gene (bacA). We demonstrate that PAISt is able to excise in modules by recombination of one internal and two flanking palindromic direct repeats. The gene intSt located at the 3( end of PAISt encodes a tyrosine recombinase. Site-specific recombination activity of intSt was tested and confirmed by heterologous expression in Streptomyces coelicolor. Comparative analysis of PAISt homologues in Streptomyces scabies 87-22 and Streptomyces acidiscabies 84-104 indicates that these islands have been fixed by sequence erosion of intSt and the recombination sites.

Figure 1. Schematic representation of the PAISt in S. turgidiscabies Car8.

Copies of the 3′ end of the bacitracin resistance gene (bacA) delimit the element in two modules of 105 Kb and 569 Kb. The virulence genes nec1 and tomA are located in the first module and the fasciation (fas) and thaxtomin (txt) biosynthetic clusters are located in the second module. The putative integrase (intSt) is located at the 3′ end of the island. The 8 bp palidromic repeats are shown within the bacA gene and its truncated copies.

Figure 2. Schematic representation of the excision of circular structures from PAISt in S. turgidiscabies Car8.

(a) PAISt and the location of the primers used for detection of specific recombination events within the att sites. Arrows indicate the locations of primers, and primer sequences are provided in Table 2. (b) Three types of excision resulting from recombination events at the att sites, which were detected using nested PCR. Excision type 1 produces CS674, excision type 2 produces CS105 and excision type 3 produces CS569.

Figure 3. Amplification of the attB and attP sequences generated after the three types of excision from PAISt.

Products were detected by nested PCR using primer pairs described in Table 2 and Figure 2. (a) PCR results resolved after electrophoresis in a 1% agarose gel. Lanes 1 to 3 show amplification of the att sites in the PAISt: attL (445 bp), attI (642 bp), and attR (341 bp). Lanes 4 to 6 show amplification of the attP sites in the circular structures (CS674, CS105 and CS569): recombination between attR and attL (attP-RxL, 380 bp), recombination between attL and attI (attP-LxI,663 bp), and recombination between attR and attI (attP-RxI, 359 bp). Lanes 7 to 9 shows amplification of the attB sites in the S. turgidiscabies chromosome: recombination between attR and attL (attB-RxL, 406 bp), recombination between attL and attI (attB-LxI, 424 bp), and recombination between attR and attI (attB-RxI, 624 bp). Lane M contains the 1 Kb ladder. (b) Sequences of the att sites with the palidromic sequences indicated in black. The flanking regions are presented in color to show recombination events within att sites (attL in green, attI in blue and attR in red).

Figure 4. Sequence analysis and structure of intSt.

(a) Amino acid alignment of INTSt with characterized integrases illustrating the three conserved regions. Conserved amino acids are indicated in grey. Asterisks indicate the amino acids R209, R375, and Y407. (b) Structural modeling of INTSt shows the residues RRY of the predicted catalytic site.

Figure 5. Integration activity of INTSt is affected by mutation in the catalytic core and deletions in the att sites.

Frequencies of transconjugants obtained when pIJintSt was mobilized into S. coelicolor A3(2). Frequency of integration was calculated based on the number of hygromycin-resistant colonies (y-axis). Point mutations in the putative catalytic core of INTSt reduces integration events. Deletion of the att sites in the plasmid pIJintStatt(−) or in the chromosome of S. coelicolor ΔbacA (pIJintSt_in_Sco att-), abolishes integration events. Plasmid pIJamp001 was used to control for illegitimate recombination (recombination not mediated by INTSt). The data shown are the means of three independent experiments. Three colonies of S. coelicolor were selected and grown to obtain spore stocks. Spore stocks were tested with the integrative plasmids as described in material and methods. Means of the integration frequencies were plotted with error bars indicating the distribution of the data.

Figure 6. Detection of multimeric integration of pIJintSt in S. coelicolor A3(2).

(a) Southern blot analysis shows three S. coelicolor A3(2) transconjugants (Sct). The blot was probed with a PCR product of intSt. Two hybridization signals are observed (1.5 Kb and 6.2 Kb). Sc indicates genomic DNA of S. coelicolor A3(2) wild type used as a control. Sizes corresponding to a 1 kb ladder are shown to the left of the southern blot. (b) PCR detection of the multimeric version of pIJintSt in S. coelicolor A3(2) transconjugants. Lane M contains the 100 bp DNA ladder. Lane 5J contains the junction formed at the 5′ end of the integration (detected with primers b1 and p2). Lane 3J contains the junction formed at the 3′ end of the integration (detected with primers b2 and p1). Lane DI contains the double integration event (detected with primers p1 and p2). Lane C is the DNA from the wild-type strain (negative control of the PCR reaction). (c) Detected events of pIJintSt integration in S. coelicolor A3(2) chromosome: figures 1 and 2 describe the integration at and duplication of the att site within bacA; figure 3 shows a second integration and the formation of a multimeric version of pIJintSt. Small black arrows indicate primers used to detect each event (Table 2). The predicted PCR product sizes are indicated. The KpnI restriction sites are indicated and correspond to the Southern blot results provided in panel a.

Figure 7. Integration of plasmids containing att sites into the chromosome of S. turgidiscabies Car8.

(a) pIJattR+ integrated at bacA in the S. turgidiscabies Car8 chromosome. Primers used to detect integration events and their products are shown (b) Amplification of the integration junctions. Lane M contains the 1 kb DNA ladder. Lanes 1 and 2 contain the products of the left junction (amplified with primers bSF-piR). Lanes 3 and 4 contain the products of the right junction (amplified with primers piF-bsR). Car8pIJattR+ is the transconjugant and Car8WT is the S. turgidicabies Car8 wild type used as a control. (c) Sequences of PCR products. The 8 bp palindrome and the flanking sequences of each PCR product are aligned.

Figure 8. Relationships of PAISt with PAISs1 in S. scabies 87–22 and PAISa1 in S. acidiscabies 84–104.

Islands PAISs1 in S. scabies and PAISa1 in S. acidiscabies, are integrated at the bacA 3′ end and contain a remnant of the intSt (red line arrow) delimited by a degenerate version of the 8 bp palindrome, (mutated residues are in red). Both islands are identical to the 105 Kb module of PAISt. The thaxtomin biosynthesis cluster (txt) is conserved in S. scabies and S. acidiscabies but is not linked to the 105 Kb island.

Figure 9. Remnants of intSt exist in PAISt, PAISs1 and PAISa1.

Alignment of INTSt with three frame translations of the 3′ end of the first 105 Kb module of PAISt and with the 3′ ends of PAISs1 and PAISa1 reveal INTSt remnants. Remnants are shown as truncated red arrows. Conserved amino acids residues and their positions in the alignment are indicated.